This study provides new insights into bat diversity and ecology in high-mountain environments of southern Europe, focusing on the Valbona River Valley in the Albanian Alps, a recognised biodiversity hotspot that remains poorly studied, despite its significant natural value. Using mist-netting and acoustic monitoring conducted between 2021 and 2025 across an altitudinal gradient, we found 24 species of bats, including 13 species previously unreported from the region. Bat assemblages varied with elevation: species richness and activity peaked at the valley floor (700-800 m a.s.l.) and mid-altitude sites (1000-1700 m a.s.l.), whereas high-altitude habitats (>2000 m a.s.l.) exhibited lower activity but higher diversity (S-W index 2.223 comparing to 1.574) due to a more even species distribution. A strong male bias across all studied sites provides robust evidence for vertical sex segregation, extending previous published data by demonstrating its persistence above 2000 m a.s.l. in the Mediterranean region. High-altitude caves use was severely limited, likely due to microclimatic constraints, indicating that such sites do not function as summer roosts. Differences in species composition between sites highlight the importance of habitat structure, particularly woody vegetation and water bodies, in shaping bat communities. Species-specific observations reveal greater ecological plasticity than previously recognised and suggest that high-altitude habitats are used opportunistically for foraging and swarming, probably influenced by cave entrance exposure to sunlight. These results demonstrate that alpine environments, although suboptimal, play a previously underestimated role in bat spatial ecology and provide essential baseline data for conservation planning under increasing anthropogenic pressure.